Diterpenes Specially Produced by Fungi: Structures, Biological Activities, and Biosynthesis (2010–2020)

Fungi have traditionally been a very rewarding source of biologically active natural products, while diterpenoids from fungi, such as the cyathane-type diterpenoids from Cyathus and Hericium sp., the fusicoccane-type diterpenoids from Fusicoccum and Alternaria sp., the guanacastane-type diterpenoids from Coprinus and Cercospora sp., and the harziene-type diterpenoids from Trichoderma sp., often represent unique carbon skeletons as well as diverse biological functions. The abundances of novel skeletons, biological activities, and biosynthetic pathways present new opportunities for drug discovery, genome mining, and enzymology. In addition, diterpenoids peculiar to fungi also reveal the possibility of differing biological evolution, although they have similar biosynthetic pathways. In this review, we provide an overview about the structures, biological activities, evolution, organic synthesis, and biosynthesis of diterpenoids that have been specially produced by fungi from 2010 to 2020. We hope this review provides timely illumination and beneficial guidance for future research works of scholars who are interested in this area.


Introduction
Fungi are widely distributed in terrestrial environments, freshwater, and marine habitats; more than one million distinctive fungal species exist, but only approximately 100,000 of these have been classified [1]. These eukaryotic microbes produce specialized metabolites that participate in a variety of ecological functions, such as quorum sensing, chemical defense, allelopathy, and maintenance of symbiotic interactions [2]. There are more than 40,000 terpenoid compounds in nature, which compose the largest family of natural products [3]. Terpenoids exist in all domains of life, but are particularly prevalent in plants, fungi, and marine invertebrates, and are essential constituents of secondary metabolism [3,4].
Diterpenoids are a class of C20 compounds derived from isoprenoid precursor geranylgeranyl diphosphate (GGPP) under the catalysis of diterpene synthases (DTSs) [5][6][7][8][9][10][11]. Prenyltransferase (PT) and terpene synthase (TPS) are key enzymes in the formation of the basic carbon skeletons of terpenoids [8,12]. The PT enzymes determine the prenyl carbon chain length, whereas the TPS enzymes generate the structural complexity of the molecular scaffolds, forming various ring structures [8]. Fungi are among the most important microbial resources for drug discovery, owing to their capability to produce structurally diverse and biologically important secondary metabolites [13,14]. It is also well known that fungi possess extraordinary biosynthetic gene clusters that may encode highly diverse biosynthetic pathways of natural products [15][16][17][18].
Between 2010 and 2020, about 400 fungal-specific diterpenes have been reported. In addition to 172 cyathane diterpenes reviewed by Bailly et al. [19] and Gao et al. [20], a total of 232 diterpenes were collected in this review (Chart 1). These diterpenoids are mainly tricyclic or tetracyclic skeletal structures such as cyathane-type, fusicoccane-type, In particular, literature investigation of known databases such as PubMed and Web of Science was conducted from 2010 to July 2020 using the keywords "diterpenes/diterpenoids" paired with "fungi", "fungal diterpenoids" paired with "structure elucidation", or "fungal diterpenoids" paired with "biosynthesis". There were no language restrictions imposed. The references were further scrutinized and, finally, 210 references were selected. The data inclusion criteria included: (1) diterpenes/diterpenoids isolated from fungi, (2) carbon skeleton obtained only from fungi or rarely from other sources, (3) studies on the biological activities of diterpenes/diterpenoids and their derivatives that had been carried out in vitro or in vivo, (4) studies on the biosynthesis of diterpenes/diterpenoids and their derivatives. The data exclusion criteria included: (1) carbon skeleton of diterpenes/diterpenoids obtained in abundance from other sources, such as plants, bacteria and so on, (2) duplication of data and titles and/or abstracts not meeting the inclusion criteria. Cyathane diterpenes are a group of natural products that possess unusual, angularly fused 5/6/7 tricyclic cores, and they are characteristic of certain basidiomycete species including Cyathus, Hericium, and Sarcodon ( Figure 1). For example, there have been more than 170 compounds isolated from fungi such as Cyathus africanus and Hericium erinaceus [19,20,41]. These compounds have a common biosynthetic precursor and can be produced Since the ITS sequence of Sarcodon cyrneus was not available, Sarcodon sp. (MK049936.1) was selected, since it is in the same family with S. cyrneus. The evolutionary history was inferred by using the maximum likelihood method and the general time reversible model [62]. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed [63]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [63]. Initial tree(s) for the heuristic search were obtained automatically by applying the Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.2219)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 0.00% sites). This analysis involved 20 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + noncoding. There were 924 positions in the final dataset. The evolutionary analysis was conducted in MEGA X (version 10.2.2) [64].

Cyathane
To understand the source genera of cyathane diterpenoids, we performed a phylogenetic analysis by using the maximum likelihood method and the general time re-versible model [62][63][64] for all the species involved in the reviews by Bailly et al. [19] and Gao et al. [20]. The results show that source genera are grouped based on their regiospecificity, i.e., genera Cyathus, Hericium, and Sarcodon were clustered into different clades ( Figure 1). Taxonomically, Cyathus africanus, C. hookeri, C. gansuensis, C. subglobisporus, C. stercoreus, and C. striatus all belonged to the genus Cyathus. They were close to each other, and first, they gathered into one branch, then, they gathered into one branch with Strobilurus tenacellus of the genus Strobilurus, and finally gathered into one branch with other genera (Figure 1). C. earlei and C. helenae also belonged to the genus Cyathus, they were close to each other, and first, they gathered into one branch, then, they gathered into one branch with Gerronema albidum of the genus Gerronema. Similarly, Hericium erinaceus, H. flagellum, and Hericium sp. WBSP8, Sarcodon scabrosus, S. glaucopus, and other species were close to each other. Existing studies have shown that most fungal metabolites are encoded by biosynthetic gene clusters (BGCs) [17]. The natural product BGCs of species in the same genus tend to be highly homologous, and BGC functional divergence gives rise to the evolution of new secondary metabolites, indicating that species-level sampling in these three genera for natural products mining will yield significant returns for cyathane diterpenoids discovery.

Cyclopiane
To understand the source genera of cyathane diterpenoids, we performed a phyl netic analysis by using the maximum likelihood method and the general time rever model [62][63][64] for all the species involved in the reviews by Bailly et al. [19] and Gao e [20]. The results show that source genera are grouped based on their regiospecificity genera Cyathus, Hericium, and Sarcodon were clustered into different clades (Figur Taxonomically, Cyathus africanus, C. hookeri, C. gansuensis, C. subglobisporus, C. sterco and C. striatus all belonged to the genus Cyathus. They were close to each other, and they gathered into one branch, then, they gathered into one branch with Strobil tenacellus of the genus Strobilurus, and finally gathered into one branch with other ge (Figure 1). C. earlei and C. helenae also belonged to the genus Cyathus, they were clos each other, and first, they gathered into one branch, then, they gathered into one bra with Gerronema albidum of the genus Gerronema. Similarly, Hericium erinaceus, H. flagel and Hericium sp. WBSP8, Sarcodon scabrosus, S. glaucopus, and other species were clos each other. Existing studies have shown that most fungal metabolites are encoded biosynthetic gene clusters (BGCs) [17]. The natural product BGCs of species in the s genus tend to be highly homologous, and BGC functional divergence gives rise to evolution of new secondary metabolites, indicating that species-level sampling in t three genera for natural products mining will yield significant returns for cyath diterpenoids discovery.

Cyclopiane
Cyclopiane diterpenoids comprise a class of tetracyclic diterpenes with unique s folds. They are characterized by a highly fused 6/5/5/5 ring system. The structural v tions of cyclopiane diterpenoids are mainly owing to oxidation occurring at various to generate hydroxy groups [65]. In general, cyclopiane diterpenoids have mainly b isolated from different species of the genus Penicillium ( Figure 2) and have been class into two groups according to the functionality at C-1, i.e., conidiogenols and conidi nones. The former featured with a hydroxy group at C-1, while the later possessed a bonyl group at C-1 [66]. Specifically, Penicillium commune MCCC 3A00940, P. sp. F23sp. YPGA11, P. cyclopium, P. roqueforti IFM 48062, P. sp. TJ403-2, P. chrysogenum QENand Leptosphaeria sp. XL026 have been reported to produce conidiogenol-type dite noids, while P. commune MCCC 3A00940, P. chrysogenum MT-12, P. sp. YPGA11, an cyclopium have been reported to produce conidiogenone-type diterpenoids (Figur Structurally, cyclopiane diterpenoids differ from the aberrarane-type diterpenoid abe rone, which has shown in vitro antimalarial activity against a chloroquine-resistant st of the protozoan parasite Plasmodium falciparum isolated from the Caribbean sea w Pseudopterogorgia elisabethae [67]. The molecular structure of aberrarone was establis by spectral analysis and subsequently confirmed by X-ray crystallographic analysis. S cyclopiane compounds exhibited pronounced biological activities, such as conidiatio duction, cytotoxic, anti-inflammatory, antimicrobial, and antiallergic effects. Cyclopiane diterpenoids comprise a class of tetracyclic diterpenes with unique scaffolds. They are characterized by a highly fused 6/5/5/5 ring system. The structural variations of cyclopiane diterpenoids are mainly owing to oxidation occurring at various sites to generate hydroxy groups [65]. In general, cyclopiane diterpenoids have mainly been isolated from different species of the genus Penicillium ( Figure 2) and have been classified into two groups according to the functionality at C-1, i.e., conidiogenols and conidiogenones. The former featured with a hydroxy group at C-1, while the later possessed a carbonyl group at C-1 [66]. Specifically, Penicillium commune MCCC 3A00940, P. sp. F23-2, P. sp. YPGA11, P. cyclopium, P. roqueforti IFM 48062, P. sp. TJ403-2, P. chrysogenum QEN-24S, and Leptosphaeria sp. XL026 have been reported to produce conidiogenol-type diterpenoids, while P. commune MCCC 3A00940, P. chrysogenum MT-12, P. sp. YPGA11, and P. cyclopium have been reported to produce conidiogenone-type diterpenoids ( Figure 2). Structurally, cyclopiane diterpenoids differ from the aberrarane-type diterpenoid aberrarone, which has shown in vitro antimalarial activity against a chloroquine-resistant strain of the protozoan parasite Plasmodium falciparum isolated from the Caribbean sea whip Pseudopterogorgia elisabethae [67]. The molecular structure of aberrarone was established by spectral analysis and subsequently confirmed by X-ray crystallographic analysis. Some cyclopiane compounds exhibited pronounced biological activities, such as conidiation induction, cytotoxic, anti-inflammatory, antimicrobial, and antiallergic effects.

Conidiogenol Type
Conidiogenol 1 is a potent and selective inducer of conidiogenesis in the liquid culture of Penicillium cyclopium under non-nutrient limiting conditions [66]. Conidiogenol B 2 has been obtained from the deep-sea derived fungus P. commune MCCC 3A00940 [68]. Conidiogenols C 3 and D 4 have been isolated from a deep-sea derived fungus P. sp. YPGA11 [65].
The absolute structure of cyclopiane diterpenoids was first confirmed by Abe and coworkers, in 2018, with the aid of the crystal sponge method [69]. Using the genome-mining approach, a chimeric enzyme of prenyltransferase-diterpene synthase (PT-TS) discovered from P. chrysogenum MT-12 was designated as P. chrysogenum cyclopiane-type diterpene synthase (PcCS). The new diterpene alcohol metabolite 5 was produced after the gene heterologously expressed in Aspergillus oryzae, and the crystalline sponge method also revealed the absolute configuration of 5 [69]. The PT domain of PcCS first generated geranylgeranyl diphosphate (GGPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) (Scheme 1A). Then, GGPP was converted into 5 by a cyclization reaction catalyzed by the TS domain of PcCS (Scheme 1B).

Conidiogenol Type
Conidiogenol 1 is a potent and selective inducer of conidiogenesis in the liquid culture of Penicillium cyclopium under non-nutrient limiting conditions [66]. Conidiogenol B 2 has been obtained from the deep-sea derived fungus P. commune MCCC 3A00940 [68]. Conidiogenols C 3 and D 4 have been isolated from a deep-sea derived fungus P. sp. YPGA11 [65].
The absolute structure of cyclopiane diterpenoids was first confirmed by Abe and coworkers, in 2018, with the aid of the crystal sponge method [69]. Using the genome-mining approach, a chimeric enzyme of prenyltransferase-diterpene synthase (PT-TS) discovered from P. chrysogenum MT-12 was designated as P. chrysogenum cyclopiane-type diterpene synthase (PcCS). The new diterpene alcohol metabolite 5 was produced after the gene heterologously expressed in Aspergillus oryzae, and the crystalline sponge method also revealed the absolute configuration of 5 [69]. The PT domain of PcCS first generated geranylgeranyl diphosphate (GGPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) (Scheme 1A). Then, GGPP was converted into 5 by a cyclization reaction catalyzed by the TS domain of PcCS (Scheme 1B).

Conidiogenone Type
Conidiogenone 6, first isolated from Penicillium cyclopium, was also an inducer of conidiation [66,70]. The biosynthetic pathway of (-)-conidiogenone 6 has been fully elucidated by the heterologous expression of biosynthetic genes in Aspergillus oryzae and by in vitro enzyme assay with 13 C-labeled substrates [71]. After construction of deoxyconidiogenol by the action of bifunctional terpene synthases (PchDS gene obtained from Penicillium chrysogenum, and PrDS gene identified from Penicillium roqueforti showed significant homology to PchDS), one cytochrome P450 catalyzed two rounds of oxidation to furnish conidiogenone 6. The cyclization mechanism catalyzed by terpene synthase, involving successive 1,2-alkyl shifts, was fully elucidated using 13 C-labeled geranylgeranyl pyrophosphate (GGPP) as a substrate (Scheme 2). Scheme 2. Proposed cyclization mechanism catalyzed by PchDS/PrDS [71].
Three new cyclopiane diterpenes 13β-hydroxy conidiogenone C 18 and 12β-hydroxy conidiogenones C 19 and D 20 have been isolated and identified from a sea sedimentderived fungus Penicillium sp. TJ403-2 [75]. Their absolute configurations were further established by X-ray crystallography experiment. Compounds 18-20 were evaluated for their anti-inflammatory activity against LPS-induced NO production, and compound 18 showed notable inhibitory potency with an IC 50 value of 2.19 µM, which was three-fold lower than the positive control indomethacin (IC 50 8.76 µM). Further Western blot and immunofluorescence experiments demonstrated that 18 inhibited the NF-κB-activated pathway.
Leptosphin C 21 has been isolated from the solid cultures of an endophytic fungus Leptosphaeria sp. XL026 [76]. Its structure was elucidated by extensive spectroscopic methods and single-crystal X-ray diffraction.
Brassicicenes Q-X 51-58 have been isolated from the phytopathogenic fungus Alternaria brassicicola [99]. Brassicicene S 53 was found to show significant anti-inflammatory activity against the production of NO, TNF-α, and IL-1β at 10 μM. Further Western blot and immunofluorescence experiments found the mechanism of 53 inhibiting the NF-κBactivated pathway.

Biosynthesis of Fusicoccane Diterpenes
A unique chimeric enzyme PaFS, possessing both a geranylgeranyl diphosphate (GGDP) synthase domain and a diterpene cyclase domain, has been identified from Phomopsis amygdali [109]. A biosynthetic gene cluster of brassicicene C 36, a fusicoccadiene synthase (AbFS) containing 11 genes (orf1 to orf11, Scheme 5A), has been identified in Alternaria brassicicola ATCC 96836 from genome database search [110,111]. In vivo and in vitro studies have clearly revealed the function of Orf8 and Orf6 as a fusicoccadiene synthase similar to PaFS and methyltransferase, respectively. In this gene cluster, five genes (orf1, orf2, orf5, orf7, and orf11) encoded cytochrome P450s. Orf9 was a key dioxygenase to determine the aglycon structures of fusicoccin and brassicicene [112].
Other fusicoccane-type diterpene synthases have been identified from bacteria or fungus, such as CotB2 from bacteria responsible for the biosynthesis of cyclooctat-9-en-7-ol 84 [113], and SdnA from fungus responsible for the biosynthesis of cycloaraneosene 85 [114]. The same 5/8/5 tricyclic skeleton occurred in the sesterterpene ophiobolin F for which the terpene synthase AcOS has been reported from Aspergillus clavatus [115]. Oikawa and co-workers applied the Aspergillus oryzae heterologous expression system to functionally characterize cryptic bifunctional terpene synthase genes found in fungal genomes and identified the sesterfisherol (contains a characteristic 5/6/8/5 tetracyclic system) synthase gene (NfSS) from Neosartorya fischeri [116]. A unique P450 enzyme bscF has been identified in the phytopathogen Pseudocercospora fijiensis that generated two structurally different products from the single substrate. In addition to the heterologous expression of the eight genes, bscA-bscH elucidated the biosynthetic pathway for brassicicenes (Scheme 5B) [117].
A new fusicoccane-type diterpene synthase MgMS has been identified from the fungus Myrothecium graminearum by the genome mining method, which catalyzed the formation of the new diterpene alcohol myrothec-15(17)-en-7-ol 86 with all the seven stereocenters being introduced in the cyclization steps and conserved in the structure of the product. Based on this, its novel cyclization mode was unambiguously assigned (Scheme 6) [118].
Heptemerones A-G 103-109 have been isolated from cultures of Coprinus heptemerus [137,138]. Radianspenes A-M 110-122 have been obtained from Coprinus radians [139]. Among the biological activities of these isolates, the inhibition of fungal germination was the most potent, and depended highly on the composition of the assay medium [137]. Radianspene C 112 showed inhibitory activity against human breast carcinoma (MDA-MB-435) cell with an IC50 value of 0.91 μM [139]. Investigation of secondary metabolites from the fungal Coprinus plicatilis led to the discovery of several new guanacastane-type diterpenoids, named plicatilisins A-D 123-126 [140] and E-H 127-130 [141]. In vitro cytotoxic activities against the human cancer cell lines (HepG2, HeLa, MDA-MB-231, BGC-823, HCT 116, and U2OS) showed that plicatilisin A 123 exhibited significant cytotoxicity with IC50 values ranging from 1.2 to 6.0 μM [140].
Eleven new guanacastane-type diterpenoids dahlianes A-K 142-152 have been obtained from the fungus Verticillium dahlia that was isolated from the gut of insect Coridius chinensis [144,145]. In the cytotoxicity evaluation against human tumor cell lines, dahlianes B 143 and C 144 exhibited significant cytotoxicity against human breast cancer cell MCF-7 with IC50 values of 3.35 and 4.72 μM, respectively [144]. In addition, the isolates were evaluated for their cytotoxicity toward drug-sensitive and DOX resistant MCF-7 cells by MTT assay. As a result, dahliane G 148 showed an 80-fold potentiation effect on the sensitization of doxorubicin at the concentration of 15 μM when screening the reversal activity on doxorubicin-resistant human breast cancer cell (MCF-7/DOX) [145].
Eleven new guanacastane-type diterpenoids dahlianes A-K 142-152 have been obtained from the fungus Verticillium dahlia that was isolated from the gut of insect Coridius chinensis [144,145]. In the cytotoxicity evaluation against human tumor cell lines, dahlianes B 143 and C 144 exhibited significant cytotoxicity against human breast cancer cell MCF-7 with IC 50 values of 3.35 and 4.72 µM, respectively [144]. In addition, the isolates were evaluated for their cytotoxicity toward drug-sensitive and DOX resistant MCF-7 cells by MTT assay. As a result, dahliane G 148 showed an 80-fold potentiation effect on the sensitization of doxorubicin at the concentration of 15 µM when screening the reversal activity on doxorubicin-resistant human breast cancer cell (MCF-7/DOX) [145].

Harziene
Harziene is a small group of diterpenoids that have a unique 4/7/5/6 tetracyclic scaffold. They have mainly been obtained from different Trichoderma species and rarely from liverworts [147]. Harziandione 157 was the first harziene diterpenoid isolated from the

Harziene
Harziene is a small group of diterpenoids that have a unique 4/7/5/6 tetracyclic scaffold. They have mainly been obtained from different Trichoderma species and rarely from liverworts [147]. Harziandione 157 was the first harziene diterpenoid isolated from the liquid culture of T. harzianum, in 1992 [148]. Harzianone 158, a new harziene diterpene, has been isolated from an alga-endophytic isolate of T. longibrachiatum [149]. The structure with absolute configuration of 158 was unambiguously identified by NMR and mass spectrometric methods as well as quantum chemical calculations. In addition, the absolute configuration of harziandione 157 was supported by optical rotation calculation, and the structure of isoharziandione isolated from culture filtrate of a strain of Trichoderma viride [150] was revised to harziandione 157 on the basis of 13 C NMR data comparison and calculation.

Harziene
Harziene is a small group of diterpenoids that have a unique 4/7/5/6 tetracyclic scaffold. They have mainly been obtained from different Trichoderma species and rarely from liverworts [147]. Harziandione 157 was the first harziene diterpenoid isolated from the liquid culture of T. harzianum, in 1992 [148]. Harzianone 158, a new harziene diterpene, has been isolated from an alga-endophytic isolate of T. longibrachiatum [149]. The structure with absolute configuration of 158 was unambiguously identified by NMR and mass spectrometric methods as well as quantum chemical calculations. In addition, the absolute configuration of harziandione 157 was supported by optical rotation calculation, and the structure of isoharziandione isolated from culture filtrate of a strain of Trichoderma viride [150] was revised to harziandione 157 on the basis of 13 C NMR data comparison and calculation.
The terpene cyclization mechanism of harzianone 158 has been studied by feeding experiments using selectively 13 C-and 2 H-labeled synthetic mevalonolactone isotopologues, followed by the analysis of the incorporation patterns of 13 C NMR spectroscopy and GC/MS, and the structure of harzianone 158 was further supported from a 13 C-13 C COSY experiment of the in vivo generated fully 13 C-labeled diterpenoid (Scheme 7) [151].
Four new harziene-related compounds 159−162 have been isolated from an endophytic fungus Trichoderma atroviridae UB-LMA [152]. Among them, 159 is a potential derivative of geranylgeranyl diphosphate and may represent the biosynthetic precursor of this scarce family of compounds (Scheme 7). Recently, the first total synthesis of nominal harziene diterpenoid 160 has been achieved; stereochemical analysis and subsequent synthesis of the epimeric tertiary alcohol led to the reassignment of configuration for compound 160 as shown for harzianol I 180 [153].
The terpene cyclization mechanism of harzianone 158 has been studied by feeding experiments using selectively 13 C-and 2 H-labeled synthetic mevalonolactone isotopologues, followed by the analysis of the incorporation patterns of 13 C NMR spectroscopy and GC/MS, and the structure of harzianone 158 was further supported from a 13 C-13 C COSY experiment of the in vivo generated fully 13 C-labeled diterpenoid (Scheme 7) [151].
Four new harziene-related compounds 159-162 have been isolated from an endophytic fungus Trichoderma atroviridae UB-LMA [152]. Among them, 159 is a potential derivative of geranylgeranyl diphosphate and may represent the biosynthetic precursor of this scarce family of compounds (Scheme 7). Recently, the first total synthesis of nominal harziene diterpenoid 160 has been achieved; stereochemical analysis and subsequent synthesis of the epimeric tertiary alcohol led to the reassignment of configuration for compound 160 as shown for harzianol I 180 [153]. Scheme 7. Biosynthetic mechanism to harziene and taxadiene scaffolds [151,152].
Two new harziene diterpene lactones, i.e., harzianelactones A 169 and B 170, and five new ones, i.e., harzianones A-D 171-174 and harziane 175, have been isolated from the soft coral-derived fungus Trichoderma harzianum XS-20090075 [159]. These compounds exhibited potent phytotoxicity against seedling growth of amaranth and lettuce. Harzianone E 176, which exhibited weak antibacterial activity against Photobacterium angustum, has been obtained from the culture of coral-derived fungus T. harzianum treated with 10 µM sodium butyrate [160]. Harzianols F-J 177-181 and three known derivatives have been obtained from the liquid fermentation of an endophytic fungus T. atroviride B7 [161]. Among them, compound 180 exhibited significant antibacterial effect against Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus with EC 50 values of 7.7, 7.7, and 9.9 µg/mL, respectively. Meanwhile, cytotoxic activity of 180 against three cancer cell lines was also observed [161].

Pleuromutilin
Pleuromutilin 192 is a diterpene with a tricyclic skeleton possessing antimicr properties. It was first discovered from two basidiomycete fungal species including rotus mutilis (synonymous to Clitopilus scyphoides f. mutilus) and Pleurotus passeckeri (synonymous to Clitopilus passeckerianus) [168], and then produced by a number of o related species [169]. Its chemical structure and cyclisation mechanism has been e dated by independent works [170][171][172], while total synthesis has been achieved [173,174]. The semi-synthetic pleuromutilin analogues tiamulin 193 and valnemulin have been used for over three decades to treat economically important infections in s and poultry without showing any significant development of resistance in their ta bacteria [21][22][23][24][25]. In recent years, extensive research including structure-activity rela

Pleuromutilin
Pleuromutilin 192 is a diterpene with a tricyclic skeleton possessing antimicrobial properties. It was first discovered from two basidiomycete fungal species including Pleurotus mutilis (synonymous to Clitopilus scyphoides f. mutilus) and Pleurotus passeckerianus (synonymous to Clitopilus passeckerianus) [168], and then produced by a number of other related species [169]. Its chemical structure and cyclisation mechanism has been elucidated by independent works [170][171][172], while total synthesis has been achieved by [173,174]. The semi-synthetic pleuromutilin analogues tiamulin 193 and valnemulin 194 have been used for over three decades to treat economically important infections in swine and poultry without showing any significant development of resistance in their target bacteria [21][22][23][24][25]. In recent years, extensive research including structure-activity relationship studies have been conducted to generate new orally available pleuromutilin derivatives having been used systemically in human medicine to treat acute bacterial skin and skin structure infections, as well as multidrug-resistant tuberculosis [175][176][177][178].
The gene cluster for the antibiotic pleuromutilin 192 has been isolated in Clitopilus passeckerianus [179]. Total de novo biosynthesis of pleuromutilin 192 was achieved through the expression of the entire gene cluster in the secondary host Aspergillus oryzae, proving that the seven genes isolated were sufficient for biosynthesis of the diterpene antibiotic. Heterologous expression of genes from the pleuromutilin gene cluster in A. oryzae revealed the biosynthesis of the antibiotic pleuromutilin 192 (Scheme 10) and generated bioactive semi-synthetic derivatives [180].
Four new tetraquinane diterpenoids crinipellins E-H 209-212 have been isolated from fermentations of a Crinipellis species [197]. Crinipellins E-G 209-211 inhibited the LPS/IFNγ induced CXCL10 promoter activity in transiently transfected human MonoMac6 cell in a dose-dependent manner with IC 50 values of 15, 1.5, and 3.15 µM, respectively. Moreover, crinipellins E-G 209-211 reduced mRNA level and synthesis of proinflammatory mediators such as cytokines and chemokines in LPS/IFN-γ stimulated MonoMac6 cell.

Psathyrin
Two skeletally novel tetracyclic diterpenoids that possess a novel 5/5/4/6 tetracyclic system, psathyrins A 216 and B 217, have been characterized from cultures of the basidiomycete Psathyrella candolleana. They displayed weak antibacterial activities against Staphylococcus aureus and Salmonella enterica. The biosynthetic pathway of 216 and 217 was proposed to start from GGPP and the final products were obtained through a series of reactions (Scheme 12) [201].

Psathyrin
Two skeletally novel tetracyclic diterpenoids that possess a novel 5/5/4/6 tetracyclic system, psathyrins A 216 and B 217, have been characterized from cultures of the basidiomycete Psathyrella candolleana. They displayed weak antibacterial activities against Staphylococcus aureus and Salmonella enterica. The biosynthetic pathway of 216 and 217 was proposed to start from GGPP and the final products were obtained through a series of reactions (Scheme 12) [201].

Psathyrin
Two skeletally novel tetracyclic diterpenoids that possess a novel 5/5/4/6 tetracyclic system, psathyrins A 216 and B 217, have been characterized from cultures of the basidiomycete Psathyrella candolleana. They displayed weak antibacterial activities against Staphylococcus aureus and Salmonella enterica. The biosynthetic pathway of 216 and 217 was proposed to start from GGPP and the final products were obtained through a series of reactions (Scheme 12) [201].

Eryngiolide
Eryngiolide A 223 has been isolated from the solid culture of the edible mushroom Pleurotus eryngii [203]. It is the first member of diterpenoids with a novel skeleton deriving from a cyclododecane core fused with two γ-lactone units [203]. It has exhibited moderate cytotoxicity against two human cancer cell lines (Hela and HepG2) in vitro. Biogenetically, eryngiolide A 223 could be the first diterpene not synthesized from GGPP unit, which indicated a completely new route for diterpene biosynthesis in nature (Scheme 13).

Trichodermanin
Trichodermanin A 224, a structurally unique diterpenoid with skeletal carbons arranged compactly in a 6/5/6/6 ring system, has been isolated from cultures of Trichoderma atroviride [204]. Its absolute configuration was elucidated by single crystal X-ray diffraction. Wickerols A 225 and B 226 were two novel diterpenoids produced by Trichoderma atroviride and the absolute configuration of 226 was confirmed by X-ray crystallographic analysis [205,206]. Wickerol A 225 showed potent antiviral activity against the A/H1N1 flu virus (A/PR/8/34 and A/WSN/33 strains) with an IC50 value of 0.07 μg/mL, but not active against the A/H3N2 virus. Wickerol B 226 also showed anti-influenza virus activity against A/PR/8/34 virus with an IC50 value of 5.0 μg/mL [206].

Eryngiolide
Eryngiolide A 223 has been isolated from the solid culture of the edible mushroom Pleurotus eryngii [203]. It is the first member of diterpenoids with a novel skeleton deriving from a cyclododecane core fused with two γ-lactone units [203]. It has exhibited moderate cytotoxicity against two human cancer cell lines (Hela and HepG2) in vitro. Biogenetically, eryngiolide A 223 could be the first diterpene not synthesized from GGPP unit, which indicated a completely new route for diterpene biosynthesis in nature (Scheme 13).

Trichodermanin
Trichodermanin A 224, a structurally unique diterpenoid with skeletal carbons arranged compactly in a 6/5/6/6 ring system, has been isolated from cultures of Trichoderma atroviride [204]. Its absolute configuration was elucidated by single crystal X-ray diffraction. Wickerols A 225 and B 226 were two novel diterpenoids produced by Trichoderma atroviride and the absolute configuration of 226 was confirmed by X-ray crystallographic analysis [205,206]. Wickerol A 225 showed potent antiviral activity against the A/H1N1 flu virus (A/PR/8/34 and A/WSN/33 strains) with an IC 50 value of 0.07 µg/mL, but not active against the A/H3N2 virus. Wickerol B 226 also showed anti-influenza virus activity against A/PR/8/34 virus with an IC 50 value of 5.0 µg/mL [206].

Trichodermanin
Trichodermanin A 224, a structurally unique diterpenoid with skeletal carbons arranged compactly in a 6/5/6/6 ring system, has been isolated from cultures of Trichoderma atroviride [204]. Its absolute configuration was elucidated by single crystal X-ray diffraction. Wickerols A 225 and B 226 were two novel diterpenoids produced by Trichoderma atroviride and the absolute configuration of 226 was confirmed by X-ray crystallographic analysis [205,206]. Wickerol A 225 showed potent antiviral activity against the A/H1N1 flu virus (A/PR/8/34 and A/WSN/33 strains) with an IC50 value of 0.07 μg/mL, but not active against the A/H3N2 virus. Wickerol B 226 also showed anti-influenza virus activity against A/PR/8/34 virus with an IC50 value of 5.0 μg/mL [206]. 1.
The new skeleton of wickerols A 225 and B 226 was revealed by the feeding experiments of [1-13 C]-, [2-13 C]-, and [1,2-13 C2]acetates, respectively [206]. The cyclization mechanism of wickerol B 226 was predicted, as shown in Scheme 14. First, pyrophosphate was ejected from the terminus of the boat-like transition state of GGPP, forming a verticillen-12-yl cation intermediate, the same as the first step of phomactatriene and taxadiene biosynthesis [207]. 1,2-Rearrangements of β-methyl and α-hydride occurred at the six-membered ring part, then, the ring inversion and cyclization progressed to form the 6/5/9 ring intermediate.
The new skeleton of wickerols A 225 and B 226 was revealed by the feeding experiments of [1-13 C]-, [2-13 C]-, and [1,2-13 C 2 ]-acetates, respectively [206]. The cyclization mechanism of wickerol B 226 was predicted, as shown in Scheme 14. First, pyrophosphate was ejected from the terminus of the boat-like transition state of GGPP, forming a verticillen-12-yl cation intermediate, the same as the first step of phomactatriene and taxadiene biosynthesis [207]. 1,2-Rearrangements of β-methyl and α-hydride occurred at the six-membered ring part, then, the ring inversion and cyclization progressed to form the 6/5/9 ring intermediate. A rearrangement proceeded to expand the ring from five to six membered, and the last step resulted in the formation of the 6/5/6/6 ring skeleton. The C-8 position of wickerol A 225 was oxidized by a cytochrome P450 to give wickerol B 226. Scheme 14. Incorporation patterns of [1-13 C]-, [2-13 C]-, and [1,2-13 C 2 ]-acetates enriched wickerol B 226, and proposed mechanism of cyclization from GGPP to wickerols [206].

Conclusions and Future Prospects
Diterpenoids show huge potential for drug discovery and development due to their extensive biological functions and structural diversity. Fungal diterpenoids are a diverse family of hybrid natural products with potent bioactivities and intriguing structural architectures. A large number of fungal diterpenoids have exhibited significant anti-inflammatory, cytotoxic, anti-MRSA, antimicrobial, antiviral, antihypertensive, and platelet aggregationinhibitory activities. Consequently, these bioactive diterpenoids are always hot trending topics for the synthesis community [173,174,186]. Nevertheless, the structural complexity and limited availability of natural products remain obstacles to synthesizing a large collection of natural products and their structural analogues in sufficient amounts. Thus, a synthetic biology method based on the combination of heterologous biosynthesis and genome mining is a promising approach to translate enormous amounts of biosynthetic gene information to richly diverse natural products. Interestingly, while fungi have evolved their systems to create terpenoid diversity, they have also biosynthesized some of the same classes of terpenoids found in plants, bacteria, and other organisms. These relationships provide accessible and renewable prokaryotic systems for eukaryotic natural product biosynthesis and enzymology. In conclusion, we hope it is evident from this review that most of the fungal diterpenoids are biologically active with a few key scaffolds paving a path towards potential drug discovery and development.

Conflicts of Interest:
The authors declare no conflict of interest.